Fluorescent sensors for imaging of interstitial calcium

Calcium in interstitial fluids is central to systemic physiology and a crucial ion pool for entry into cells through numerous plasma membrane channels. Its study has been limited by the scarcity of methods that allow monitoring in tight inter-cell spaces of living tissues. Here we present high performance ultra-low affinity genetically encoded calcium biosensors named GreenT-ECs. GreenT-ECs combine large fluorescence changes upon calcium binding and binding affinities (Kds) ranging from 0.8 mM to 2.9 mM, making them tuned to calcium concentrations in extracellular organismal fluids. We validated GreenT-ECs in rodent hippocampal neurons and transgenic zebrafish in vivo, where the sensors enabled monitoring homeostatic regulation of tissue interstitial calcium. GreenT-ECs may become useful for recording very large calcium transients and for imaging calcium homeostasis in inter-cell structures in live tissues and organisms.


Parental protein and linker optimizations
The starting parental construct for GreenT-EC was obtained by replacing tryptophan 148 in mNeonGreen with the minimal calcium binding domain of Troponin C (TnC) previously optimized during the design of the FRET-based "Twitch" family of indicators 1 .The four amino acid long linkers flanking the minimal domain in Twitch-2B were initially also incorporated for generating our mNeonGreen-based indicator (Supplementary Fig. 1).Twitch-2B was crystalized 2 , which provided further insights into the role of the linkers, which were not only limited to provide flexibility for an optimal orientation change between the bound and apo forms of of purified proteins from 100% to 600-700 %, with a Kd of 127 nM and an extinction coefficient (EC) of 67 mM -1 cm -1 in the calcium bound state.The second evolutionary step was obtained using this variant to further optimize linker 1 (sequence change: GADA to LGWD at position 148-151), reaching a maximum fluorescence change of 1100 % after calcium binding.The Kd of this variant was 221 nM and the EC was 75 mM -1 cm -1 .We also evaluated the effect of linker extensions by adding 3-7 extra amino acids to the linkers but no improvements were observed.

Region 1
We finally identified a critical region which had an eminent effect on the indicator response (sequence CRS at position 225-227) within the coding region of mNeonGreen immediately after the second linker.We named this region R1 (Supplementary Fig. 1a, b).After screening this region, we selected two main next generation variants: a variant with a single mutation C225N (named NRS) and one with a triple mutation changing the amino acids CRS to RTT (named RTT).Both variants had fluorescence changes of around 6000% after binding calcium.Interestingly, the excitation and emission maxima shifted from values of 508/517 nm in mNeonGreen to 497/515 nm in NRS and 504/515 nm in RTT.In addition, while the Kd of both variants remained in the range 135-200 nM, the calcium bound states had high ECs (116 mM -1 cm -1 for RTT and 100 mM -1 cm -1 for NRS).

Random mutagenesis
We next opened two different evolutionary branches, starting from either NRS or RTT and kept on mutagenizing and screening both parental constructs further using random mutagenesis procedures.In particular, the mutation F241L showed a positive impact in the response at the cost of a small reduction of brightness.This region was also critical for the kinetics, with the mutation of F241Y leading to faster offkinetics at the expenses of a reduced EC.Interestingly, the side chain of this amino acid is directed towards the chromophore inside the β-barrel, indicating a role of the internal network of interactions in the chromophore transitions induced for calcium binding.We stablished that the best performances were obtained with RTT F241L and NRS F241Y.At this stage of evolution, we had achieved high responses both with purified proteins (> 7000 %) with extinction coefficients corresponding to 80-90% of the values for the original mNeonGreen protein.However, we detected a reduced expression level of these variants in mammalian cells and bacteria compared to control indicators.We therefore focused the efforts at increasing the expression levels, folding and solubility, while monitoring carefully that the brightness, response and calcium binding affinity remained in the desired range.This was achieved by performing several rounds of random mutagenesis screenings in bacterial plates and further testing of selected variants in mammalian cells.During these steps, around 130000 colonies were evaluated.The general strategy consisted in identifying mutations leading to increases in expression and further evaluation of the impact of these modifications in the performance of the indicator.If an amino acid position showed interesting effects on other parameters besides expression, site saturation mutagenesis and further screenings were performed on those positions.We used different strategies to generate variability by means of PCR: i) Using degenerate primers in only one amino acid position, ii) combining 2-5 primers containing specific identified mutations for different amino acids or iii) including multiple degenerated primers, each one corresponding to one amino acid position.In many cases, we found mutations that led to strong increases in the expression levels without any apparent effect on the properties measured on purified proteins, but completely abolished the response in mammalian cells.One of these cases was the mutation M100V, whose side chain is directed inside the beta barrel.This highlighted the need of evaluating at every step the selected candidates in mammalian cells.Some of the amino acid positions that were individually evaluated by site directed mutagenesis were A11, T37, W61, M100, A146, C159, D198, M215, K229, F241, S255, T246, R258 and M277.

Crystal structure
During the mentioned screenings, we expressed and characterized more than 300 variants, generating a detailed map of the effect that different amino acids had on the indicator performance, and identified several indicators with interesting properties derived from both the NRS and RTT parental variants.We then aimed to obtain a crystal structure that could allow us to rationalize the information obtained during the screenings and further evolve the variants.After several attempts of crystallization, we were successful with one variant (Named NRS 1.2) derived from the NRS F241Y parental construct that contained the extra mutations A11G/T37S/R258I (Fig. 1a, Supplementary Fig. 1).The incorporation of these 3 mutations led to a 10-fold increase in expression levels without deteriorating the response, brightness or Kd of the parental construct.
In addition, we removed the last nine C-terminus amino acids (VMGMDELYK) to reduce the presence of flexible regions during the crystallization.The crystal structure (Supplementary Fig. 1) showed many interesting functional features of the indicator.As was also observed in the case of Twitch-2B, a residue of linker 2, that in our case was Leu221, stablish strong interactions with a hydrophobic cluster in TnC (Supplementary Fig. 1a).In addition, multiple amino acids of both linkers, TnC and mNeonGreen establish polar and hydrophobic interactions in these regions (Supplementary Fig. 1b).Importantly, Leu148 (first amino acid of linker 1) is positioned in close proximity to the chromophore, likely playing an important role in stabilizing the closed conformation of the β-barrel and the anionic form of the chromophore (Supplementary Fig. 1c).Another critical residue pointed by the obtained structure is Asn225.This amino acid (originally a cysteine in mNeonGreen) acts as a bridge between the calcium binding-domain and the mNeonGreen β-barrel structure (Supplementary Fig. 1d) establishing clear polar interactions with both domains.Finally, aligning the indicator with the published structure of mNeonGreen (PDB 5ltr), it becomes evident that the insertion of the calcium binding domain in the β-strand containing amino acid residues 135-145 of mNeonGreen induces a higher degree the flexibility and thus a large conformational change not only in the mentioned strand but also in the region consisting of amino acid residues 190-200 (Supplementary Fig. 1e).Finally, we also observe a significant change in the position of the chromophore (Supplementary Fig. 1f) that might explain the shift in the excitation maximum from 507 to 497 nm characteristic of the NRS variants.It is interesting to mention that most of the mutations increasing solubility, folding and expression were located in mNeonGreen and with a few exceptions, their side chains were pointing outside the βbarrel.

Developing ultra-low affinity variants
At this point, we had obtained different indicators evidencing about 80-90% of the brightness of mNeonGreen in the calcium bound state, a high response in solution and appropriate expression levels.
However, the calcium binding affinity was typically in the range of 200 nM, far too high for any interstitial calcium measurements.In this regard, the last step was to test mutations previously described in TnC with the aim of reducing the binding affinity 1 .We found two insertions in the calcium binding EF-hand loops (169D+ and 205S+) being the most effective at drastically reducing the binding affinity with a minor impact in the protein response and brightness.While inserting these residues in the NRS templates led to variants with dissociation constants in the µM range, the incorporation of the same mutations in the RTT derivatives, led to the desired mM range in the calcium binding affinities.We selected a candidate containing the mentioned A11G/T37S/R258I mutations, plus G248D, that also led to higher expression levels without affecting any other parameter.The insertion of 169D and 205S led to the variants GreenT-EC and GreenT-EC.brespectively.Including the additional amino acid exchange T260I, discovered separately, in GreenT-EC.b,further reduced the binding affinity to generate GreenT-EC.c.
the indicator, but also established key interactions with the TnC domain and the fluorescent proteins to stabilize the conformations.The parental version of mNeonGreen-TnC had a maximal fluorescence change of about 100 %.It was subsequently subjected to extensive saturation mutagenesis in both linker regions randomizing 4 amino acid positions at the time.The first notable improvement was achieved during the optimization of linker 2 (sequence change: PIYP to LTDN at position 221-224) and brought the response

Data collection and refinement statistics.
Statistics for the highest-resolution shell are shown in parentheses.